This invention relates to novel pyridylpyrrole compounds, processes for the preparation thereof, the use thereof in treating cytokines mediated diseases and/or cell adhesion molecules mediated diseases, and pharmaceutical compositions for use in such therapy.
Cytokines possess a multitude of regulatory and inflammatory effects. Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) are biological substances produced by a variety of cells, such as monocytes or macrophages. IL-1 has been demonstrated to mediate a variety of biological activities thought to be important in immunoregulation and other physiological conditions such as inflammation.
There are many disease states in which excessive or unregulated IL-1 production is implicated in exacerbating and/or causing the disease. These include rheumatoid arthritis (RA), osteoarthritis (OA), endotoxemia and/or toxic shock syndrome, other acute and chronic inflammatory disease states such as the inflammatory reaction induced by endotoxin or inflammatory bowel disease (IBD), tuberculosis, atherosclerosis, muscle degeneration, cachexia, psoriatic arthritis, Reiter""s syndrome, gout, traumatic arthritis, rubella arthritis and acute synovitis. Recent evidence also links IL-1 activity to diabetes.
Excessive or unregulated TNF production has been implicated in mediating or exacerbating a number of diseases including RA, rheumatoid spondylitis, OA, gouty arthritis and other arthritic conditions; sepsis, septic shock, endotoxin shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome (ARDS), cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcoisosis, bone resorption diseases, reperfision injury, graft vs. host reaction, allograft rejections, fever and myalgias due to infection such as influenza, cachexia secondary to infection or malignancy, cachexia, secondary to acquired immune deficiency syndrome (AIDS), ARC (AIDS related complex), keloid formation, scar tissue formation, Crohn""s disease, ulcerative colitis or pyresis. The concept of anti-TNF therapy has been validated by the demonstration that soluble TNF receptor and neutralizing monoclonal antibodies (MAbs) against TNF showed therapeutic efficacy in a variety of preclinical and clinical studies (e.g., Elliott, M. J. et al., The Lancet, 1994, 344, 1125. Dullemen, H. M. V. et al., Gastroenterology, 1995, 109, 129.)
Interleukin-8 (IL-8) is a chemotactic factor first identified and characterized in 1987. IL-8 is produced by several cell types including neutrophils, mononuclear cells, fibroblasts, endothelial cells, epithelial cells and keratinocytes. Elevated IL-8 levels have been reported in joint fluids in RA, gouty arthritis, psoriatic scale and ARDS. Its production from endothelial cells is induced by IL-1, TNF or lipopolysachharide (LPS). IL-8 has been shown to have chemoattractant properties for neutrophils, T-lymphocytes and basophils. In addition, it promote angiogenesis as well as neutrophil activation, including lysozomal enzyme release and respiratory burst. IL-8 has also been shown to increase the surface expression of Mac-1 (CD 11b/CD18) on neutrophils, this may contribute to increased adhesion of the neutrophils to vascular endothelial cells. Many diseases are characterized by massive neutrophil infiltration. Conditions associated with an increased IL-8 production would benefit by compounds which are suppressive of IL-8 production.
IL-1 and TNF affect a wide variety of cells and tissues, and these cytokines as well as other leukocyte derived cytokines are important and critical inflammatory mediators of a wide variety of disease states and conditions. The inhibition of these cytokines is of benefit in controlling, reducing and alleviating many of these disease states.
Cellular movement and adhesion are a fundamental biological response to external stimuli. During an inflammatory response, leukocytes must leave the plasma compartment and migrate to the point of antigenic insult. The mechanism of this migratory event is a complex interplay between soluble mediators and membrane-bound cellular adhesion molecules. Soluble cellular chemotactic factors, which are produced in the damaged tissue by a variety of resident cells, set up a chemical concentration gradient out to the plasma compartment. Interaction of these factors with their receptors on leukocytes leads to a directional migration of the leukocytes toward increasing concentrations of the chemotactic factor. Simultaneously, various adhesion molecules are upregulated on the leukocyte which mediate the initial rolling on the endothelial tissue, binding to a specific ligand on the activated endothelial tissue, and finally migration between endothelial cells into the tissue. The steps in this cascade of events are mediated by the interaction of specific cell surface protein, termed xe2x80x9ccell adhesion molecules (CAMs)xe2x80x9d. E-selectin (ELAM-1, endothelial leukocyte adhesion molecule-1), ICAM-1 (intercellular adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1) are three major adhesion molecules whose expression on endothelial cells is upregulated upon treatment with inflammatory stimuli. ICAM-1 is expressed at low levels on resting endothelium and is markedly induced in response to cytokines such as IL-1, TNF and interferon-xcex3 (IFN-xcex3). VCAM-1 is not expressed in resting endothelium but is induced by IL-1, TNF and IL-4. Induction of both ICAM-1 and VCAM-1 occurs 4 to 6 hours after cytokine treatment and cell surface expression remains elevated for up to 72 hours after treatment with cytokines. On the other hand, induction of transcription of the E-selectin gene by cytokines such as IL-1 and TNF results in an increase in the expression on the surface of endothelial cells peaking approximately 4-6 hours after challenge, and returns toward a basal level of expression by 24 hours.
The concept of anti-CAMs therapy has been validated by the demonstration that MAbs against ICAM-1 and antisense oligonucleotide against ICAM-1 showed therapeutic efficacy in a variety of preclinical and clinical studies (A. F. Kavanaugh et al., Arthritis Rhetun, 1994, 37, 992; C. E. Haug et al., Transplantation, 1993, 55, 766; and J. E. Jr. Sligh et al., Proc. Natl. Acad. Sci., 1993, 90, 8529). Further support comes from the reports of the in vivo activity of sLeX and related carbohydrates, antagonists of E-selectin mediated adhesion (M. S. Mulligan et al., Nature, 1993, 364, 149-151). Thus, the potential therapeutic targets for CAMs inhibitors range from, but are not limited to, RA, IBD and psoriasis to ischemia/reperfusion injury, autoimmune diabetes, organ transplantation, ARDS, tumor metastases and AIDS, as is evident from the many ongoing development activities. The regulation of the functions of CAMs is of benefit in controlling, reduction and alleviating many of these disease states. There remains a need for treatment, in this field, for compounds which are capable of inhibiting cytokines production and/or CAMs expression. The pyridylpyrroles of the present invention have been shown in an in vitro assay to inhibit cytokines production and/or CAMs expression.
International Publication No. WO 95/18122 discloses 2-heteroaryl-3-cyanopyrrole compounds having agrochemical activities.
British Patent No. GB1311336 discloses quaternary salts of pyrrolylpyridine compounds (e.g., 4,4xe2x80x2-(3,4-dimethylpyrrol-2,5-diyl)bis(1-n-heptylpyridinium bromide) having antibacterial and fungal properties.
The present invention provides a compound of the formula: 
and its pharmaceutically acceptable salts, wherein
R1 is selected from the following:
(a) hydrogen, R6xe2x80x94, R6xe2x80x94NHxe2x80x94, hydroxy-R6xe2x80x94 or R6xe2x80x94Oxe2x80x94R6xe2x80x94;
(b) R6xe2x80x94COxe2x80x94, R6xe2x80x94Oxe2x80x94COxe2x80x94R6xe2x80x94, carboxy-R6xe2x80x94, NH2xe2x80x94COxe2x80x94 or R6xe2x80x94NHxe2x80x94COxe2x80x94; and
(c) Arxe2x80x94, Arxe2x80x94R6xe2x80x94, Arxe2x80x94NHxe2x80x94or Arxe2x80x94COxe2x80x94;
wherein Ar is selected from phenyl, naphthyl, pyridyl, quinolyl, thienyl, furyl, pyrrolyl, indolyl, benzothienyl and benzofuryl, the aryl or heteroaryl groups being optionally substituted with one or two substituents selected from C1-4 alkyl, C1-4 alkoxy, halo-substituted C1-4 alkyl, halo-substituted C1-4 alkoxy, nitro, hydroxy, amino, R6xe2x80x94NHxe2x80x94, (R6)2Nxe2x80x94, halo, formyl, halo-substituted phenoxy, halo-substituted phenyl, C1-4 alkyl-substituted phenoxy, halo-substituted phenylthio, C1-4 alkoxycarbonyl, C1-4 alkylthio and C1-4 alkyl-SOxe2x80x94; and
wherein R6 is C1-6alkyl optionally substituted by up to four halogen atoms;
R2 and R4 are independently selected from the following:
(d) hydrogen, halo, R6xe2x80x94, C2-6 alkenyl, C2-6 alkynyl, hydroxy-R6xe2x80x94, R6xe2x80x94Oxe2x80x94R6xe2x80x94, mercapto-R6xe2x80x94, R6xe2x80x94Sxe2x80x94R6xe2x80x94, xe2x80x94NH2, R6xe2x80x94NHxe2x80x94, R6)2xe2x80x94Nxe2x80x94, R6xe2x80x94Oxe2x80x94, R6xe2x80x94Sxe2x80x94, R6xe2x80x94SOxe2x80x94 and R6xe2x80x94SO2xe2x80x94;
(e) 1,4-dioxa-8-azaspiro[4,5]-decanyl, 
wherein Y is selected from xe2x80x94NH, xe2x80x94Nxe2x80x94R6, xe2x80x94Nxe2x80x94Ar, O and S; l is 0, 1, 2, 3, 4 or 5; n is independently 0, 1 or 2; and Ar is as defined above;
(f) Arxe2x80x94, Arxe2x80x94R6xe2x80x94, Arxe2x80x94C2-6 alkenyl, Arxe2x80x94C2-6 alkynyl, Arxe2x80x94Oxe2x80x94, Arxe2x80x94Oxe2x80x94R6xe2x80x94, Arxe2x80x94R6xe2x80x94Oxe2x80x94, Arxe2x80x94Sxe2x80x94, Arxe2x80x94R6xe2x80x94Sxe2x80x94, Arxe2x80x94NHxe2x80x94, (Ar)2xe2x80x94R6xe2x80x94, Arxe2x80x94R6xe2x80x94NHxe2x80x94 or (Ar)2xe2x80x94Nxe2x80x94;
(g) R6xe2x80x94COxe2x80x94, xe2x80x94NO2, NH2xe2x80x94COxe2x80x94, R6xe2x80x94NHxe2x80x94COxe2x80x94, (R6)2xe2x80x94Nxe2x80x94COxe2x80x94, Arxe2x80x94COxe2x80x94, (Arxe2x80x94R6)2-Nxe2x80x94COxe2x80x94, Arxe2x80x94R6xe2x80x94COxe2x80x94, Arxe2x80x94NHxe2x80x94COxe2x80x94 or Arxe2x80x94R6xe2x80x94NHxe2x80x94COxe2x80x94; and
(h) R6xe2x80x94COxe2x80x94NHxe2x80x94, Arxe2x80x94COxe2x80x94NHxe2x80x94, Arxe2x80x94R6xe2x80x94COxe2x80x94NHxe2x80x94or H2Nxe2x80x94COxe2x80x94NHxe2x80x94;
wherein Ar and R6 are as defined above, provided that R2 is not Ar;
R3 is selected from the following:
(i) C2-6 alkenyl, C2-6 alkynyl, halo, hydroxy-R6xe2x80x94, R6xe2x80x94Oxe2x80x94R6xe2x80x94, R6xe2x80x94Sxe2x80x94R6xe2x80x94, Arxe2x80x94, NH2xe2x80x94R6xe2x80x94 or R6xe2x80x94NHxe2x80x94R6;
(j) formyl, carboxy, carboxy-R6xe2x80x94, tetrazolyl, triazolyl, imidazolyl, oxazolyl, thiazolyl, R6xe2x80x94COxe2x80x94, C2-6 alkenylxe2x80x94COxe2x80x94, C2-6 alkynylxe2x80x94COxe2x80x94, R6xe2x80x94COxe2x80x94R6xe2x80x94, C2-6 alkenylxe2x80x94COxe2x80x94R6xe2x80x94, C2-6alkynylxe2x80x94COxe2x80x94R6xe2x80x94, R6xe2x80x94Oxe2x80x94COxe2x80x94, R6xe2x80x94Oxe2x80x94COxe2x80x94R6xe2x80x94, R6xe2x80x94Sxe2x80x94COxe2x80x94, C2-6 alkenylxe2x80x94Oxe2x80x94COxe2x80x94, C2-6 alkynylxe2x80x94Oxe2x80x94COxe2x80x94 or R6xe2x80x94Oxe2x80x94R6xe2x80x94COxe2x80x94;
(k) R6xe2x80x94COxe2x80x94NHxe2x80x94, Arxe2x80x94COxe2x80x94NHxe2x80x94, Arxe2x80x94R6xe2x80x94COxe2x80x94NHxe2x80x94, xe2x80x94NH2, R6xe2x80x94NHxe2x80x94, (R6)2xe2x80x94Nxe2x80x94, H2Nxe2x80x94COxe2x80x94NHxe2x80x94, R6xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94, (R6)2xe2x80x94Nxe2x80x94COxe2x80x94NHxe2x80x94, Arxe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94, (Ar)2xe2x80x94Nxe2x80x94COxe2x80x94NHxe2x80x94, HOxe2x80x94Nxe2x95x90CHxe2x80x94R6xe2x80x94, R6Oxe2x80x94Nxe2x95x90CHxe2x80x94 or R6Oxe2x80x94Nxe2x95x90CHxe2x80x94R6xe2x80x94;
(l) R6xe2x80x94SOxe2x80x94, R6xe2x80x94NHxe2x80x94SO2xe2x80x94 R6xe2x80x94SO2xe2x80x94, xe2x80x94SO2NH2, xe2x80x94SONH2, R6xe2x80x94NHxe2x80x94SOxe2x80x94, Arxe2x80x94SOxe2x80x94, Arxe2x80x94R6SOxe2x80x94, Arxe2x80x94SO2xe2x80x94, C2-6 alkenyl-SO2xe2x80x94, C2-6 alkynyl-SO2xe2x80x94, Arxe2x80x94R6xe2x80x94SO2xe2x80x94, Arxe2x80x94NHxe2x80x94SO2xe2x80x94, Arxe2x80x94R6xe2x80x94NHxe2x80x94SO2xe2x80x94, Arxe2x80x94NHxe2x80x94SOxe2x80x94 or Arxe2x80x94R6xe2x80x94NHxe2x80x94SOxe2x80x94; and
(m) Arxe2x80x94COxe2x80x94, Arxe2x80x94R6xe2x80x94COxe2x80x94, Arxe2x80x94C2-6 alkenylxe2x80x94COxe2x80x94, Arxe2x80x94C2-6 alkynylxe2x80x94COxe2x80x94, Arxe2x80x94Oxe2x80x94COxe2x80x94, Arxe2x80x94Oxe2x80x94R6xe2x80x94COxe2x80x94, Arxe2x80x94Sxe2x80x94R6xe2x80x94COxe2x80x94, Arxe2x80x94R6xe2x80x94Oxe2x80x94COxe2x80x94, Arxe2x80x94R6xe2x80x94Sxe2x80x94COxe2x80x94, (Ar)2xe2x80x94C2-6 alkenyl-COxe2x80x94, (Ar)2xe2x80x94C2-6 alkynyl-COxe2x80x94, (Ar)2xe2x80x94R6xe2x80x94Oxe2x80x94COxe2x80x94 or (Ar)2xe2x80x94R6xe2x80x94Sxe2x80x94COxe2x80x94;
wherein Ar and R6 are as defined above; or
two of R2, R3 and R4 together form a group of the formula xe2x80x94A1xe2x80x94B1xe2x80x94A2xe2x80x94B2xe2x80x94A3xe2x80x94 which, together with the carbon atoms to which A1 and A3 are attached, defines a ring having 5 to 8 ring atoms, the ring optionally being substituted with one or two substituents selected from hydroxy, R6, C1-4 alkoxy and Ar, wherein A1, A2 and A3 are independently direct bond or C1-4 alkylene and B1 and B2 are independently direct bond, O, S, SO, CO, NH or NR6;
R5 is independently selected from the following:
(n) hydrogen, halo, R6xe2x80x94, hydroxy-R6xe2x80x94 or R6xe2x80x94Oxe2x80x94R6xe2x80x94;
(o) Arxe2x80x94, Arxe2x80x94R6xe2x80x94, Arxe2x80x94Oxe2x80x94, Arxe2x80x94Sxe2x80x94, Arxe2x80x94NHxe2x80x94 or Arxe2x80x94COxe2x80x94; and
(p) R6xe2x80x94COxe2x80x94, R6xe2x80x94Oxe2x80x94COxe2x80x94 or R6xe2x80x94NHxe2x80x94COxe2x80x94; or
two of R5 which are attached to adjacent carbon atoms on the pyridine ring complete a fused benzene ring, the benzene ring being optionally substituted with one or two substituents selected from C1-4 alkyl, halo-substituted C1-4 alkyl, halo-substituted C1-4 alkoxy, nitro, hydroxy, amino and halo;
wherein R6 and Ar are as defined above;
m is 0, 1, 2, 3 or 4; and
the nitrogen atom of the pyridyl ring attached to the 5-position of the pyrrole ring is optionally replaced by a N oxide group.
The present invention also provides a pharmaceutical composition for the treatment or alleviation of cytokine-mediated diseases or CAMs mediated diseases, which comprises a therapeutically effective amount of a compound of said formula (I) or its pharmaceutically acceptable carrier.
The present invention further provides a method for the treatment of disease conditions caused by cytokine-mediator or CAMs mediator, in a mammalian subject, which comprises administering to said subject a therapeutically effective amount of a compound of the formula (I).
The present invention also provides a pharmaceutical composition for the treatment or alleviation of cytokine-mediated diseases or CAMs mediated diseases, which comprises a therapeutically effective amount of a compound of the formula (I):
and its pharmaceutically acceptable salts, wherein
R1 is selected from the following:
(a) hydrogen, R6xe2x80x94, R6xe2x80x94NHxe2x80x94, hydroxy-R6xe2x80x94 or R6xe2x80x94Oxe2x80x94R6xe2x80x94;
(b) R6xe2x80x94COxe2x80x94, R6xe2x80x94Oxe2x80x94COxe2x80x94R6xe2x80x94, carboxy-R6xe2x80x94, NH2xe2x80x94COxe2x80x94 or R6xe2x80x94NHxe2x80x94COxe2x80x94; and
(c) Arxe2x80x94, Arxe2x80x94R6xe2x80x94, Arxe2x80x94NHxe2x80x94 or Arxe2x80x94COxe2x80x94;
wherein Ar is selected from phenyl, naphthyl, pyridyl, quinolyl, thienyl, furyl, pyrrolyl, indolyl, benzothienyl and benzofuryl, the aryl or heteroaryl groups being optionally substituted with one or two substituents selected from C1-4 alkyl, C1-4 alkoxy, halo-substituted C1-4 alkyl, halo-substituted C1-4 alkoxy, nitro, hydroxy, amino, R6xe2x80x94NHxe2x80x94, (R6)2Nxe2x80x94, halo, formyl, halo-substituted phenoxy, halo-substituted phenyl, C1-4 alkyl-substituted phenoxy, halo-substituted phenylthio, C1-4 alkoxycarbonyl, C1-4 alkylthio and C1-4 alkyl-SOxe2x80x94; and
wherein R6 is C1-6 alkyl optionally substituted by up to four halogen atoms;
R2 and R4 are independently selected from the following:
(d) hydrogen, halo, R6xe2x80x94, C2-6 alkenyl, C2-6 alkynyl, hydroxy-R6xe2x80x94, R6xe2x80x94Oxe2x80x94R6xe2x80x94, mercapto-6xe2x80x94, R6xe2x80x94Sxe2x80x94R6xe2x80x94, xe2x80x94NH2, R6xe2x80x94NHxe2x80x94, (R6)2xe2x80x94Nxe2x80x94, R6xe2x80x94Oxe2x80x94, R6xe2x80x94Sxe2x80x94, R6xe2x80x94SOxe2x80x94and R6xe2x80x94SO2;
(e) 1,4-dioxa-8-azaspiro[4,5]-decanyl, 
wherein Y is selected from xe2x80x94NH, xe2x80x94Nxe2x80x94R6, xe2x80x94Nxe2x80x94Ar, O and S; l is 0, 1, 2, 3, 4 or 5; n is independently 0, 1 or 2; and Ar is as defined above;
(f) Arxe2x80x94, Arxe2x80x94R6xe2x80x94, Arxe2x80x94C2-6 alkenyl, Arxe2x80x94C2-6 alkynyl, Arxe2x80x94Oxe2x80x94, Arxe2x80x94Oxe2x80x94R6xe2x80x94, Arxe2x80x94R6xe2x80x94Oxe2x80x94, Arxe2x80x94Sxe2x80x94, Arxe2x80x94R6xe2x80x94Sxe2x80x94, Arxe2x80x94NHxe2x80x94, (Ar)2xe2x80x94R6xe2x80x94, Arxe2x80x94R6xe2x80x94NHxe2x80x94 or (Ar)2xe2x80x94Nxe2x80x94;
(g) R6xe2x80x94COxe2x80x94, xe2x80x94NO2, NH2xe2x80x94COxe2x80x94, R6xe2x80x94NHxe2x80x94COxe2x80x94, (R6)2xe2x80x94Nxe2x80x94COxe2x80x94, Arxe2x80x94COxe2x80x94, (Arxe2x80x94R6)2xe2x80x94Nxe2x80x94COxe2x80x94, Arxe2x80x94R6xe2x80x94COxe2x80x94, Arxe2x80x94NHxe2x80x94COxe2x80x94 or Arxe2x80x94R6xe2x80x94NHxe2x80x94COxe2x80x94; and
(h) R6xe2x80x94COxe2x80x94NHxe2x80x94, Arxe2x80x94COxe2x80x94NHxe2x80x94, Arxe2x80x94R6xe2x80x94COxe2x80x94NHxe2x80x94or H2Nxe2x80x94COxe2x80x94NHxe2x80x94;
wherein Ar and R6 are as defined above, provided that R2 is not Ar;
R3 is selected from the following:
(i) C2-6 alkenyl, C2-6 alkynyl, halo, hydroxy-R6xe2x80x94, R6xe2x80x94Oxe2x80x94R6xe2x80x94, R6xe2x80x94Sxe2x80x94R6xe2x80x94, Arxe2x80x94, NH2R6xe2x80x94 or R6NHxe2x80x94R6;
(j) cyano, H2Nxe2x80x94COxe2x80x94, formyl, carboxy, carboxy-R6xe2x80x94, tetrazolyl, triazolyl, imidazolyl, oxazolyl, thiazolyl, R6xe2x80x94COxe2x80x94, C2-6 alkenyl-COxe2x80x94, C2-6 alkynyl-COxe2x80x94, R6xe2x80x94COxe2x80x94R6xe2x80x94, C2-6 alkenyl-COxe2x80x94R6xe2x80x94, C2-6 alkynyl-COxe2x80x94R6xe2x80x94, R6xe2x80x94Oxe2x80x94COxe2x80x94, R6xe2x80x94COxe2x80x94R6xe2x80x94, R6xe2x80x94Sxe2x80x94COxe2x80x94, C2-6 alkenylxe2x80x94Oxe2x80x94COxe2x80x94, C2-6 alkynylxe2x80x94Oxe2x80x94COxe2x80x94 or R6xe2x80x94Oxe2x80x94R6xe2x80x94COxe2x80x94;
(k) R6xe2x80x94COxe2x80x94NHxe2x80x94, Arxe2x80x94COxe2x80x94NHxe2x80x94, Arxe2x80x94R6xe2x80x94COxe2x80x94NHxe2x80x94, xe2x80x94NH2, R6xe2x80x94NHxe2x80x94, (R6)2xe2x80x94Nxe2x80x94, H2Nxe2x80x94COxe2x80x94NHxe2x80x94, R6xe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94, (R6)xe2x80x94Nxe2x80x94COxe2x80x94NHxe2x80x94, Arxe2x80x94NHxe2x80x94COxe2x80x94NHxe2x80x94, (Ar)2xe2x80x94Nxe2x80x94COxe2x80x94NHxe2x80x94, HOxe2x80x94Nxe2x95x90CHxe2x80x94R6xe2x80x94, R6Oxe2x80x94Nxe2x95x90CHxe2x80x94 or R6Oxe2x80x94Nxe2x95x90CHxe2x80x94R6xe2x80x94;
(l) R6xe2x80x94SOxe2x80x94, R6xe2x80x94NHxe2x80x94SO2xe2x80x94R6xe2x80x94SO2xe2x80x94, xe2x80x94SO2NH2, xe2x80x94SONH2, R6xe2x80x94NHxe2x80x94SOxe2x80x94, Arxe2x80x94SOxe2x80x94, Arxe2x80x94R6xe2x80x94SOxe2x80x94, Arxe2x80x94SO2xe2x80x94, C2-6 alkenyl-SO2xe2x80x94, C2-6 alkynyl-SO2xe2x80x94, Arxe2x80x94R6xe2x80x94SO2xe2x80x94, Arxe2x80x94NHxe2x80x94SO2xe2x80x94, NHxe2x80x94SO2xe2x80x94, Arxe2x80x94NHxe2x80x94SOxe2x80x94 or Arxe2x80x94R6xe2x80x94NHxe2x80x94SOxe2x80x94; and
(m) Arxe2x80x94COxe2x80x94, Arxe2x80x94R6xe2x80x94COxe2x80x94, Arxe2x80x94C2-6 alkenyl-COxe2x80x94, Arxe2x80x94C2-6 alkynyl-COxe2x80x94, Arxe2x80x94Oxe2x80x94COxe2x80x94, Arxe2x80x94Oxe2x80x94R6xe2x80x94COxe2x80x94, Arxe2x80x94Sxe2x80x94R6xe2x80x94COxe2x80x94, Arxe2x80x94R6xe2x80x94Oxe2x80x94COxe2x80x94, Arxe2x80x94R6xe2x80x94Sxe2x80x94COxe2x80x94, (Ar)2xe2x80x94C2-6 alkenyl-COxe2x80x94, (Ar)2xe2x80x94C2-6 alkynyl-COxe2x80x94, (Ar)2xe2x80x94R6xe2x80x94Oxe2x80x94COxe2x80x94 or (Ar)2xe2x80x94R6xe2x80x94Sxe2x80x94COxe2x80x94;
wherein Ar and R6 are as defined above; or
two of R2, R3 and R4 together form a group of the formula xe2x80x94A1xe2x80x94B1xe2x80x94A2xe2x80x94B2xe2x80x94A3xe2x80x94 which, together with the carbon atoms to which A1 and A3 are attached, defines a ring having 5 to 8 ring atoms, the ring optionally being substituted with one or two substituents such as hydroxy, R6, C1-4 alkoxy or Ar, wherein A1, A2 and A3 are independently direct bond or C1-4 alkylene and B1 and B2 are independently direct bond, O, S, SO, CO, NH or NR6;
R5 is independently selected from the following:
(n) hydrogen, halo, R6xe2x80x94, hydroxy-R6xe2x80x94 or R6xe2x80x94Oxe2x80x94R6xe2x80x94;
(o) Arxe2x80x94, Arxe2x80x94R6xe2x80x94, Arxe2x80x94Oxe2x80x94, Arxe2x80x94Sxe2x80x94, Arxe2x80x94NHxe2x80x94 or Arxe2x80x94COxe2x80x94; and
(p) R6xe2x80x94COxe2x80x94, R6xe2x80x94Oxe2x80x94COxe2x80x94 or R6xe2x80x94NHxe2x80x94COxe2x80x94; or
two of R5 which are attached to adjacent carbon atoms on the pyridine ring complete a fused benzene ring, the benzene ring being optionally substituted with one or two substituents selected from C1-4 alkyl, halo-substituted C1-4 alkyl, halo-substituted C1-4 alkoxy, nitro, hydroxy, amino and halo;
wherein R6 and Ar are as defined above;
m is 0, 1, 2, 3 or 4; and
the nitrogen atom of the pyridyl ring attached to the 5-position of the pyrrole ring is optionally replaced by a N oxide group.
As used herein, the term xe2x80x9cC1-6 alkylxe2x80x9d means straight or branched chain saturated radicals of 1 to 6 carbon atoms, including, but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, secondary-butyl, tertiary-butyl, and the like.
As used herein, the term xe2x80x9cC2-6 alkenylxe2x80x9d means straight or branched chain unsaturated radicals of 2 to 6 carbon atoms, including, but not limited to ethenyl, 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like.
As used herein, the term xe2x80x9chaloxe2x80x9d means fluoro, chloro, bromo and iodo.
As used herein, the term xe2x80x9cN oxide groupxe2x80x9d means one represented by the following formula: 
As used herein, the term xe2x80x9cequivalent of R2axe2x80x94C(O)xe2x80x94CH2xe2x80x94R7xe2x80x9d means compounds with similar reactivity to R2axe2x80x94C(O)xe2x80x94CH2xe2x80x94R7, or compounds which can be transformed to R2axe2x80x94C(O)xe2x80x94CH2xe2x80x94R7 in situ, such as enamine equivalent R2axe2x80x94C(NH2)xe2x95x90CHxe2x80x94R7, or enolether equivalent R2axe2x80x94C(OR3a)xe2x95x90CHxe2x80x94R7.
In the formula (I), a substituent of substituted R6 (for example, hydroxy-R6xe2x80x94, carboxy-R6xe2x80x94, R6xe2x80x94Oxe2x80x94R6xe2x80x94, etc.) may be attached to any carbon atom of the R6.
In the group xe2x80x9c(Ar)2xe2x80x94R6xe2x80x94xe2x80x9d, two of Ar may be the same or different from each other, and may be attached to a same carbon atom or different carbon atoms of R6.
A preferred group of compounds of this invention includes the compound of the formula (I) wherein R1 is selected from group (a); R2 is selected from group (d), (e) or (f), provided that R2 is not Ar; R3 is selected from groups (i), (j), (k) and (m), provided that R3 is not Ar, tetrazolyl, triazolyl, imidazolyl, oxazolyl nor thiazolyl; R4 is selected from group (d), (e) or (f); and R5 is selected from group (n); and m is 0, 1 or 2.
A more preferred group of compounds of this invention includes the compounds of formula (I) wherein R1 is hydrogen, C1-4 alkyl, C1-4 alkylamino, halo substituted C1-4 alkyl, hydroxy-C1-4 alkyl, C1-4 alkoxyalkyl or halo C1-4 alkoxy-C1-4 alkyl; R2 is hydrogen, halo, R6xe2x80x94, hydroxy-R6xe2x80x94 or R6xe2x80x94Oxe2x80x94R6xe2x80x94; R3 is C2-6 alkenyl, C2-6 alkynyl, halo, hydroxy-R6xe2x80x94, R6xe2x80x94Oxe2x80x94R6xe2x80x94, R6xe2x80x94Sxe2x80x94R6xe2x80x94, R6xe2x80x94NHxe2x80x94R6xe2x80x94, formyl, carboxy, carboxy-R6xe2x80x94, R6xe2x80x94COxe2x80x94, C2-6 alkenyl-COxe2x80x94, C24 alkynyl-COxe2x80x94, R6xe2x80x94COxe2x80x94R6xe2x80x94, C2-6 alkenyl-COxe2x80x94R6xe2x80x94, C2-6 alkynyl-COxe2x80x94R6xe2x80x94, R6xe2x80x94Oxe2x80x94COxe2x80x94, R6xe2x80x94Oxe2x80x94COxe2x80x94R6xe2x80x94, R6xe2x80x94Sxe2x80x94COxe2x80x94, C2-4 alkenylxe2x80x94Oxe2x80x94COxe2x80x94 or R6xe2x80x94Oxe2x80x94R6xe2x80x94COxe2x80x94; R4 is hydrogen, R6xe2x80x94, morpholino optionally substituted by one, two or three C1-4 alkyl or phenyl, 1-piperidinyl optionally substituted by one, two or three C1-4 alkyl or phenyl, 4-piperazinyl optionally substituted at its 1-position by C1-4 alkyl or phenyl, pyridyl, quinolyl, furyl, thienyl or pyrrolyl, phenyl, naphthyl, pyridyl, quinolyl, thienyl, furyl, pyrrolyl, indolyl, benzothienyl or benzofuryl, and wherein said phenyl, naphthyl, pyridyl, quinolyl, thienyl, furyl, pyrrolyl, indolyl, benzothienyl or benzofuryl may optionally be substituted with one or two substituents independently selected from C1-4 alkyl, C1-4 alkoxy, hydroxy, halo, formyl, C1-4 halo-substituted alkyl, halo-substituted phenoxy, halo-substituted phenylthio, C1-4 alkoxycarbonyl, C1-4 alkylthio and C1-4 alkyl-SOxe2x80x94; Rs is hydrogen, halo, C1-4 alkyl or halo substituted C1-4 alkyl; and m is 0 or 1.
A more preferred group of compounds of this invention includes the compounds of formula (I) wherein R1 is hydrogen, C1-4 alkyl or C1-4 alkoxy-C1-4 alkyl; R2 is hydrogen, halo, C1-4 alkyl optionally substituted by halo, hydroxy-C1-4 alkyl or C1-4-alkoxy-C1-4 alkyl; R3 is C2-4 alkenyl, C2-4 alkynyl, halo, hydroxy-C1-4 alkyl, C1-4 alkoxy-C1-4 alkyl, formyl, carboxy, C1-4 alkylcarbonyl, C1-4 alkylcarbonyl-C1-4 alkyl, C1-4 alkoxy-carbonyl, C1-4 alkoxycarbonyl-C1-4 alkyl, C2-4 alkenyloxycarbonyl or C1-4 alkyloxy-C1-4-alkylcarbonyl; R2 and R3 are at the 4 and 3 positions of the pyrrole ring, respectively; R4 is C1-4 alkyl, morpholino, dimethylmorpholino, 1-piperidinyl, 4-piperazinyl optionally substituted at its 1-position by C1-4 alkyl, phenyl or pyridyl, phenyl, naphthyl, pyridyl, quinolyl, thienyl, furyl or pyrrolyl, and wherein said phenyl, naphthyl, pyridyl, quinolyl, thienyl, furyl or pyrrolyl may optionally be substituted with one or two substituents independently selected from C1-4 alkyl, hydroxy, C1-4 alkoxy, halo, formyl, fluorophenoxy, methoxycarbonyl, ethoxycarbonyl, methylthio, ethylthio and methyl-SOxe2x80x94; and R5 is hydrogen or halo.
A more preferred group of compounds of this invention includes the compounds of formula (I) wherein R1 is hydrogen, C1-4 alkyl or C1-4 alkoxy-C1-4 alkyl; R2 is C1-4 alkyl optionally substituted by halo, hydroxy-C1-4 alkyl or C1-4-alkoxy-C1-4 alkyl; R3 is C2-4 alkenyl, hydroxy-C1-4 alkyl, C1-4 alkoxy-C1-4 alkyl, formyl, C1-4 alkylcarbonyl, C1-4 alkylcarbonyl-C1-4 alkyl, C1-4 alkoxy-carbonyl, C1-4 alkoxycarbonyl-C1-4 alkyl; R4 is morpholino, 1-piperidinyl, 4-phenyl-piperazin-1-yl, 1-(2-pyridyl)-piperazin4-yl, pyridyl, phenyl, naphthyl, pyrrolyl, furyl or thienyl, and wherein said pyridyl, phenyl, naphthyl, pyrrolyl, furyl or thienyl may optionally be substituted with C1-4 alkoxy, halo, formyl, 4-fluorophenoxy, methoxycarbonyl, ethoxycarbonyl or methylthio; and R5 is hydrogen.
A preferred group of compounds of this invention includes the compound of the formula (I) wherein R3 at the 3-position and R4 at the 2-position together form a group of the formula xe2x80x94A1xe2x80x94B1xe2x80x94A2xe2x80x94B2xe2x80x94A3xe2x80x94 which, together with the carbon atoms to which A1 and A3 are attached, defines a ring having 5 to 8 ring atoms, the ring optionally being substituted with one or more substituents selected from hydroxy, R6, C1-4 alkoxy and Ar, wherein A1, A2 and A3 are independently direct bond or C1-4 alkylene and B1 and B2 are independently direct bond, O, S, SO, CO, NH or NR6.
Among these, a more preferred group of compounds of this invention includes the compound of the formula (I) wherein xe2x80x94A1xe2x80x94B1xe2x80x94A2xe2x80x94B2xe2x80x94A3xe2x80x94 is selected form xe2x80x94COxe2x80x94(CH2)3xe2x80x94, xe2x80x94COxe2x80x94(CH2)2xe2x80x94, xe2x80x94COxe2x80x94CH2xe2x80x94C(CH3)2xe2x80x94CH2xe2x80x94, xe2x80x94COxe2x80x94(CH2)2xe2x80x94C(CH3)2, xe2x80x94COxe2x80x94(CH2)4xe2x80x94, xe2x80x94COxe2x80x94CH2xe2x80x94CH(CH3)xe2x80x94CH2xe2x80x94, xe2x80x94COxe2x80x94Oxe2x80x94CH(CH3)xe2x80x94CH2xe2x80x94 and xe2x80x94COxe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94.
Also, a particularly preferred compound of the invention includes the compound of formula (I) wherein R1 is hydrogen, methyl or methoxyethyl; R2 is methyl, ethyl, monofluoromethyl, difluoromethyl, trifluoromethyl, phenyl, n-propyl, isopropyl, n-bytyl, isobutyl, methoxymethyl, nitrophenyl, hydroxymethyl or pyridyl; R3 is acetyl, propanoyl, pentanoyl, ethoxycarbonyl, methoxycarbonyl, formyl, methanesulfonyl, hydroxyethyl, hydroxymethyl, benzyloxycarbonyl, allyloxycarbonyl, carboxyl, methylethoxycarbonyl, 1,1-dimethylethoxycarbonyl, propoxycarbonyl, butoxycarbonyl or methoxyethoxycarbonyl; R2 and R3 are at the 4 and 3 positions of the pyrrole ring, respectively; R4 is methyl, phenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, naphthyl, pyridyl, quinolyl, thienyl, phenyl, hydrogen, morpholino, 1-piperidinyl, (1-phenyl)-4-piperazinyl or (2-pyridyl)-4-piperazinyl; and R5 is hydrogen.
Among the compounds of the formula (I), the most preferred compound is one of the following:
3-acetyl-4-methyl-2,5-di(4-pyridyl)-1H-pyrrole;
3-acetyl-4-ethyl-2,5-di(4-pyridyl)-1H-pyrrole;
3-acetyl-2,5-di(4-pyridyl)4-trifluoromethyl-1H-pyrrole;
3-methoxycarbonyl-4-methyl-2,5-di(4-pyridyl)-1H-pyrrole;
3-acetyl-2,4-dimethyl-5-(4-pyridyl)-1H-pyrrole;
3-acetyl-4-methyl-2-phenyl-5-(4-pyridyl)-1lH-pyrrole;
3-methyl4-oxo-2-(4-pyridyl)-4,5,6,7-tetrahydro-1H-indole;
3-acetyl-2-(4-fluorophenyl)-4-methyl-5-(4-pyridyl)-1H-pyrrole;
3-acetyl-2-(2-fluorophenyl)-4-methyl-5-(4-pyridyl)-1H-pyrrole;
4-Oxo-2-(4-pyridyl)-3,6,6-trimethyl-4,5,6,7-tetrahydro-1H-indole;
3,6-Dimethyl4-oxo-2-(4-pyridyl)-4,5,6,7-tetrahydro-1H-indole;
4-Oxo-2-(4-pyridyl)-3,7,7-trimethyl-4,5,6,7-tetrahydro-1H-indole;
3-Acetyl-2-{(4-methoxycarbonyl)phenyl}-4-methyl-5-(4-pyridyl)-1H-pyrrole;
3-Acetyl-4-methyl-2-(1-piperidinyl)-5-(4-pyridyl)-1H-pyrrole;
3-Acetyl-4-methyl-2-(4-phenylpiperazin-1-yl)-5-(4-pyridyl)-1H-pyrrole;
3-Acetyl-2-(3-chloro-4-fluorophenyl)-4-methyl-5-(4-pyridyl)-1H-pyrrole;
3-Acetyl-2-(4-chlorophenyl)-4-methyl-5-(4-pyridyl)-1H-pyrrole;
3-Acetyl-2-(4-methoxyphenyl)-4-methyl-5-(4-pyridyl)-1H-pyrrole; and
3-Acetyl-4-methyl-2-(4-morpholino)-5-(4-pyridyl)-1H-pyrrole.
The present invention also provides a process for preparing a compound of the formula; 
wherein R1, R5 and m are defined in claim 1; R2a are independently hydrogen, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, Ar or Arxe2x80x94C1. alkyl; and R7 is xe2x80x94C(O)R3a, xe2x80x94C(O)OR3a, xe2x80x94CN or xe2x80x94SO2R3a, wherein R3a is hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, Ar or Arxe2x80x94C1-4 alkyl,
which comprises reacting a compound of the formula: 
xe2x80x83or its equivalent of this compound, with a compound of the formula: 
and amine R1NH2 in a reaction-inert solvent.
The compounds of the formula (I) of the present invention can be used as an active ingredient for the treatment or alleviation of asthma, arthritis, inflammatory bowel disease, sepsis, septic shock, rhinitis, inflammation of organs, AIDS, various inflammatory diseases, cardiovascular diseases, psoriasis, thrombosis, crohn""s disease, cachexia, viral infections, gout, graft vs host disease, transplant rejection and the like.
Preferred pharmaceutical composition of this invention are those of the formula (I), wherein R1 is selected from group (a); R2 is selected from group (d), (e) or (f), provided that R2 is not Ar; R3 is selected from groups (i), (j), (k) and (m), provided that R3 is not Ar, tetrazolyl, triazolyl, imidazolyl, oxazolyl nor thiazolyl; R4 is selected from group (d), (e) or (f); and R5 is selected from group (n); and m is 0, 1 or 2.
More preferred pharmaceutical composition of this invention are those of the formula (I), wherein R1 is hydrogen, C1-4 alkyl, C1-4 alkylamino, halo substituted C1-4 alkyl, hydroxy-C1-4 alkyl, C1-4 alkoxyalkyl or halo C1-4 alkoxy-C1-4 alkyl; R2 is hydrogen, halo, R6xe2x80x94, hydroxy-R6xe2x80x94 or R6Oxe2x80x94R6xe2x80x94; R3 is C2-6 alkenyl, C2-4 alkynyl, halo, hydroxy-R6xe2x80x94, R6Oxe2x80x94R6xe2x80x94, R6xe2x80x94Sxe2x80x94R6xe2x80x94, R6xe2x80x94NHxe2x80x94R6xe2x80x94, formyl, carboxy, carboxy-R6xe2x80x94, R6xe2x80x94COxe2x80x94, C2-6 alkenyl-COxe2x80x94, C2-6 alkynyl-COxe2x80x94, R6xe2x80x94COxe2x80x94R6xe2x80x94, C2-4 alkenyl-COxe2x80x94R6xe2x80x94, C2-6 alkynyl-COxe2x80x94R6xe2x80x94, R6Oxe2x80x94COxe2x80x94, R6xe2x80x94Oxe2x80x94COxe2x80x94R6xe2x80x94, R6xe2x80x94Sxe2x80x94COxe2x80x94, C2-6 alkenylxe2x80x94Oxe2x80x94COxe2x80x94 or R6Oxe2x80x94R6xe2x80x94COxe2x80x94; R4 is hydrogen, R6xe2x80x94, morpholino optionally substituted by one, two or three C1-4 alkyl or phenyl, 1-piperidinyl optionally substituted by one, two or three C1-4 alkyl or phenyl, 4-piperazinyl optionally substituted at its 1-position by phenyl or C1-4 alkyl, pyridyl, quinolyl, furyl, thienyl or pyrrolyl, phenyl, naphthyl, pyridyl, quinolyl, thienyl, furyl, pyrrolyl, indolyl, benzothienyl or benzofuryl, and wherein said phenyl, naphthyl, pyridyl, quinolyl, thienyl, furyl, pyrrolyl, indolyl, benzothienyl or benzofuryl may optionally be substituted with one or two substituents independently selected from C1-4 alkyl, C1-4 alkoxy, hydroxy, halo, formyl, C1-4 halo-substituted alkyl, halo-substituted phenoxy, halo-substituted phenylthio, C1-4 alkoxycarbonyl, C1-4 alkylthio and C1-4 alkyl-SOxe2x80x94; R5 is hydrogen, halo, C1-4 alkyl or halo substituted C1-4 alkyl; and m is 0 or 1.
Furthermore preferred pharmaceutical composition of this invention are those of the formula (I), wherein R1 is hydrogen, C1-4 alkyl or C1-4 alkoxy-C1-4 alkyl; R2 is hydrogen, halo, C1-4 alkyl optionally substituted by halo, hydroxy-C1-4 alkyl or C1-4-alkoxy-C1-4 alkyl; R3 is C2-4 alkenyl, C2-4 alkynyl, halo, hydroxy-C1-4 alkyl, C1-4 alkoxy-C1-4 alkyl, formyl, carboxy, C1-4 alkylcarbonyl, C1-4 alkylcarbonyl-C1-4 alkyl, C1-4 alkoxy-carbonyl, C1-4 alkoxycarbonyl-C1-4 alkyl, C2-4 alkenyloxycarbonyl or C1-4 alkyloxy-C1-4-alkylcarbonyl; R2 and R3 are at the 4 and 3 positions of the pyrrole ring, respectively; R4 is C1-4 alkyl, morpholino, dimethylmorpholino, 1-piperidinyl, 4-piperazinyl optionally substituted at its 1-position by C1-4 alkyl, phenyl or pyridyl, phenyl, naphthyl, pyridyl, quinolyl, thienyl, furyl or pyrrolyl, and wherein said phenyl, naphthyl, pyridyl, quinolyl, thienyl, furyl or pyrrolyl may optionally be substituted with one or two substituents independently selected from C1-4 alkyl, hydroxy, C1-4 alkoxy, halo, formyl, fluorophenoxy, methoxycarbonyl, ethoxycarbonyl, methylthio, ethylthio and methyl-SOxe2x80x94; and Rs is hydrogen or halo.
Much furthermore preferred pharmaceutical composition of this invention are those of the formula (I), wherein R1 is hydrogen, C1-4 alkyl or C1-4 alkoxy-C1-4 alkyl; R2 is C1-4 alkyl optionally substituted by halo, hydroxy-C1-4 alkyl or C1-4-alkoxy-C1-4 alkyl; R3 is C2-4 alkenyl, hydroxy-C1-4 alkyl, C1-4 alkoxy-C1-4 alkyl, formyl, C1-4 alkylcarbonyl, C1-4 alkylcarbonyl-C1-4 alkyl, C1-4 alkoxy-carbonyl, C1-4 alkoxycarbonyl-C1-4 alkyl; R4 is morpholino, 1-piperidinyl, 4-phenyl-piperazin-1-yl, 1-(2-pyridyl)-piperazin-4-yl, pyridyl, phenyl, naphthyl, pyrrolyl, furyl or thienyl, and wherein said pyridyl, phenyl, naphthyl, pyrrolyl, furyl or thienyl may optionally be substituted with C1-4 alkoxy, halo, formyl, 4-fluorophenoxy, methoxycarbonyl, ethoxycarbonyl or methylthio; and R5 is hydrogen.
Another preferred pharmaceutical composition of this invention are those of the formula (I), wherein R3 at the 3-position and R4 at the 2-position together form a group of the formula xe2x80x94A1xe2x80x94B1xe2x80x94A2xe2x80x94B2xe2x80x94A3xe2x80x94 which, together with the carbon atoms to which A1 and A3 are attached, defines a ring having 5 to 8 ring atoms, the ring optionally being substituted with one or more substituents selected from hydroxy, R6, C1-4 alkoxy and Ar, wherein A1, A2 and A3 are independently direct bond or C1-4 alkylene and B1 and B2 are independently direct bond, O, S, SO, CO, NH or NR6.
Among these, more preferred pharmaceutical composition of this invention are those of the formula (1), wherein xe2x80x94A1xe2x80x94B1xe2x80x94A2xe2x80x94B2xe2x80x94A3xe2x80x94 is selected form xe2x80x94COxe2x80x94(CH2)3xe2x80x94, xe2x80x94COxe2x80x94(CH2)2xe2x80x94, xe2x80x94COxe2x80x94CH2xe2x80x94C(CH3)2xe2x80x94CH2xe2x80x94, xe2x80x94COxe2x80x94(CH2)2xe2x80x94C(CH3)2xe2x80x94, xe2x80x94COxe2x80x94CH2xe2x80x94CH(CH3)xe2x80x94CH2xe2x80x94, xe2x80x94COxe2x80x94Oxe2x80x94CH(CH3)xe2x80x94CH2xe2x80x94 and xe2x80x94COxe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94.
Also, particularly preferred pharmaceutical composition of this invention are those of the formula (I), wherein R1 is hydrogen, methyl or methoxyethyl; R2 is methyl, ethyl, monofluoromethyl, difluoromethyl, trifluoromethyl, phenyl, n-propyl, isopropyl, n-bytyl, isobutyl, methoxymethyl, nitrophenyl, hydroxymethyl or pyridyl; R3 is acetyl, propanoyl, pentanoyl, ethoxycarbonyl, methoxycarbonyl, formyl, methanesulfonyl, hydroxyethyl, hydroxymethyl, benzyloxycarbonyl, allyloxycarbonyl, carboxyl, methylethoxycarbonyl, 1,1-dimethylethoxycarbonyl, propoxycarbonyl, butoxycarbonyl or methoxyethoxycarbonyl; R2 and R3 are at the 4 and 3 positions of the pyrrole ring, respectively; R4 is methyl, phenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, naphthyl, pyridyl, quinolyl, thienyl, phenyl, hydrogen, morpholino, 1-piperidinyl, (1-phenyl)-4-piperazinyl or (2-pyridyl)-4-piperazinyl; and R5 is hydrogen.
Preferred individual compounds of this pharmaceutical composition of this invention are:
3-acetyl-4-methyl-2,5-di(4-pyridyl)-1H-pyrrole;
3-acetyl-4-ethyl-2,5-di(4-pyridyl)-1H-pyrrole;
3-acetyl-2,5-di(4-pyridyl)-4-trifluoromethyl-1H-pyrrole;
3-methoxycarbonyl-4-methyl-2,5-di(4-pyridyl)-1H-pyrrole;
3-acetyl-2,4-dimethyl-5-(4-pyridyl)-1H-pyrrole;
3-acetyl-4-methyl-2-phenyl-5-(4-pyridyl)-1H-pyrrole;
3-methyl-4-oxo-2-(4-pyridyl)4,5,6,7-tetrahydro-1H-indole;
3-acetyl-2-(4-fluorophenyl)-4-methyl-5-(4-pyridyl)-1H-pyrrole;
3-acetyl-2-(2-fluorophenyl)-4-methyl-5-(4-pyridyl)-1H-pyrrole;
4-Oxo-2-(4-pyridyl)-3,6,6-trimethyl-4,5,6,7-tetrahydro-1H-indole;
3,6-Dimethyl-4-oxo-2-(4-pyridyl)-4,5,6,7-tetrahydro-1H-indole;
4-Oxo-2-(4-pyridyl)-3,7,7-trimethyl-4,5,6,7-tetrahydro-1H-indole;
3-Acetyl-2-{(4-methoxycarbonyl)phenyl}-4-methyl-5-(4-pyridyl)-1H-pyrrole;
3-Acetyl-4-methyl-2-(1-piperidinyl)-5-(4-pyridyl)-1H-pyrrole;
3-Acetyl-4-methyl-2-(4-phenylpiperazin-1-yl)-5-(4-pyridyl)-1H-pyrrole;
3-Acetyl-2-(3-chloro-4-fluorophenyl)-4-methyl-5-(4-pyridyl)-1H-pyrrole;
3-Acetyl-2-(4-chlorophenyl)-4-methyl-5-(4-pyridyl)-1H-pyrrole;
3-Acetyl-2-(4-methoxyphenyl)-4-methyl-5-(4-pyridyl)-1H-pyrrole; and
3-Acetyl-4-methyl-2-(4-morpholino)-5-(4-pyridyl)-1H-pyrrole.
The compounds of this invention can be prepared by a variety of synthetic routes. Representative procedures are outlined as follows.
1. Synthesis of Pyridylpyrroles by Palladium Catalyzed Cross Coupling
The compounds of formula (I) can be prepared by using the method of Stille or Suzuki (for example, Snieckus V. et al., J. Org. Chem., 1995, 60, 292, Stille, J. K. Angew. Chem. Int. Ed. Engl., 1986, 25, 508, Mitchell, M. B. et al., Tetrahedron Lett., 1991, 32, 2273, Matteson, D. S., Tetrahedron, 1989, 45, 1859). 
(wherein R is an organometallic group such as trialkylstannyl, dialkylboronyl, boric acid or zinc halide such as zinc chloride, zinc bromide or zinc iodide; R1, R2, R3, R4 and R5 are as already defined above; and X is halo such as Cl, Br or I)
As shown in Scheme 1, the pyrrole compounds (I) can be prepared by a reaction of compound (1-1) with pyrrolyl halide (1-2), in the presence of a catalyst, preferably tetrakis(triphenylphosphine)palladium or bis(triphenylphosphine)palladium(II) chloride, in the inert solvent such as benzene, toluene, xylene, tetrahydrofuran, dioxane, dimethylformamide, preferably dioxane under suitable conditions.
The reaction of trialkyl(4-pyridyl)stannane (1-1) with pyrrolyl halides (1-2) may be carried out in an inert solvent such as benzene, toluene, xylene, tetrahydrofuran, dioxane, dimethylformamide, preferably dioxane, typically in the presence of lithium chloride and a catalyst. The catalyst may be selected from those typically employed for the so-called Stille reaction (for example, tetrakis(triphenylphosphine)palladium or bis(triphenylphosphine)palladium(II) chloride). The reaction may be run at a temperature in a range from 20 to 160xc2x0 C., preferably 60 to 130xc2x0 C., for 10 minutes to 5 days, usually 30 minutes to 15 hours.
The reaction of dialkyl(4-pyridyl)borane (1-1) with pyrrolyl halides (1-2) may be carried out in an inert solvent such as benzene, toluene, tetrahydrofuran, preferably toluene, typically in the presence of a base such as potassium hydroxide, triethylamine, sodium ethoxide, sodium acetate or quaternary ammonium halide, preferably potassium hydroxide. The catalyst may be selected from those typically employed for the so-called Suzuki reaction (for example, tetrakis(triphenylphosphine)palladium or bis(triphenylphosphine)palladium(II) chloride). The reaction is run at a temperature in the range from 20 to 160xc2x0 C., preferably 60 to 130xc2x0 C. for 10 minutes to 5 days, usually 30 minutes to 15 hours.
The reaction of 4-pyridineboronic acid (1-1) with pyrrolyl halides (1-2) may be carried out in a solvent such as benzene, toluene, dimethoxyethane, dimethylformamide, preferably dimethoxyethane, typically in the presence of a base such as potassium hydroxide, triethylamine, sodium bicarbonate, preferably sodium bicarbonate, or a combination of water and above compounds, preferably water and dimethoxyethane. The catalyst may be selected from those typically employed for the so-called Suzuki reaction (for example, tetrakis(triphenylphosphine)palladium, bis(triphenylphosphine)palladium(II) chloride, or {bis(diphenylphosphino)butane}palladium(II) chloride). The reaction is run at a temperature in the range from 20 to 160xc2x0 C., usually 60 to 130xc2x0 C. for 10 minutes to 5 days, usually 30 minutes to 15 hours.
The procedures and conditions to carry out these coupling reactions are known to those in the art, and described in several technical literatures. For example, the procedures of Gronowitz, S. et al. and Snieckus, V. et al. for alkylstannanes are described in J. Het. Chem., 1990, 27, 2165, and J. Org. Chem., 1995, 60, 292; the procedure of Terashima, M. et al. for alkyl boranes, is in Heterocycles, 1984, 22, 265 and 2471, and in Chem. Pharm. Bull., 1983, 31, 4573; and the procedures of Fischer, F. C., Mitchel, M. B. et al. and McKillop, A. et al. for boric acids are in J. Red. Trav. Chim. Pays-Bays, 1965, 84, 439, Tetrahedron Lett., 1991, 32, 2273, and Tetrahedron, 1992,48,8117.
The 4-metalpyridines (1-1) (R=metal) can be prepared according to the procedure of the above literatures. The requisite pyrrolyl halides (1-2) can be prepared from the corresponding pyrroles by halogenation known in the art. The pyrroles for halogenation are either commercially available or can be prepared by using methods known in the art, for example, Hantzsch""s method, Feist""s method, Knorr""s method and Katritzky""s method (Tetrahedron, 1995, 51, 13271).
As apparent to one skilled in the art, the compound (I) can be also obtained from a reaction of the compound (1-1) wherein R is halo and the compound (1-2) wherein X is replaced by an organometallic group such as Me3Sn, Bu3Snxe2x80x94, Et2Bxe2x80x94, (HO)2Bxe2x80x94 or zinc halide. The replacement of a halogen atom by the organometallic group can be carried out by the halogen-metal exchange, followed by a reaction of appropriate reagents such as trimethyltin chloride, tributyltin chloride, diethyl methoxyborane or trimethyl borate.
2. Synthetic Methods of 2,5-Diarylpyrroles
The compounds of the formula (Ia) can be prepared by the following novel method. 
(wherein R1, R5 and m are as defined above; R2a and R3a are independently hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, Ar or Arxe2x80x94C1-4 alkyl; and R7 is an electron withdrawing group exemplified by C(O)R3a, C(O)OR3a, CN or SO2R3a.)
As shown in Scheme 2, the compounds of the formula (Ia) can be prepared from a reaction of aldehyde (2-1), 1,3-dione (2-2a) and amine (2-3) in an inert reaction solvent. For example, 3-acetyl-4-methyl-2,5-di(4-pyridyl)-1H-pyrrole (Ia) (R1xe2x95x90R5xe2x95x90H, R2a=methyl, R7=C(O)CH3) can be efficiently prepared from 4-pyridinecarboxaldehyde (2-1) (R5=H) and 2,4-pentanedione (2-2a) (R2axe2x95x90R3a=methyl) in the presence of ammonia (2-3) (R1=H) in one step. 
This reaction may be carried out in a reaction inert solvent, for example, ethanol, methanol, toluene, xylene, tetrahydrofuran, methylene chloride, preferably ethanol. However, a solvent is not always necessary. The novel reactions of this pyrrole synthesis generally proceed at a temperature in the range from xe2x88x9220 to 250xc2x0 C., preferably 0 to 150xc2x0 C., more preferably 40 to 110xc2x0 C. for 10 minutes to 3 days, usually 30 minutes to 15 hours.
The analogs of the compounds (Ia) with a variety of functional groups, such as alkylcarboxy, cyano or sulfonyl, instead of carbonyl, can be synthesized by the use of xcex2-ketoesters (2-2b), xcex2-ketonitriles (2-2c), xcex2-ketosulfones (2-2d) or their equivalents of 1,3-diones instead of the 1,3-diones (2-2a) in the above reaction. 
As the aldehydes (2-1), quinolinecarboxaldehydes (two of R5 are attached to adjacent carbon atoms on the pyridine ring to complete a fused benzene ring) can be also used in this reaction, instead of 4-pyridinecarboxaldehyde, to afford 2,5-diquinolylpyrrole compounds. Also, 2,5-diarylpyrroles having different aryl rings at the 2 and 5-positions (Ia) can be prepared by the reaction of a mixture of two kinds of arylaldehydes (2-1) with the 1,3-dione (2-2a) or their equivalent ((2-2b)-(2-2d)) and the amine (2-3). For example, 2-pyridyl-5-quinolylpyrroles can be prepared by a reaction of a mixture of 4-pyridinecarboxaldehyde (2-1) and 4-quinolinecarboxaldehyde (2-1), 1,3-dione (2-2a) or their equivalent ((2-2b)-(2-2d)) and amine (2-3). As shown in Scheme 2b, 3-acetyl-4-methyl-2-(4-pyridyl)-5-(4-quinolyl)-1H-pyrrole can be efficiently prepared from 4-pyridinecarboxaldehyde, 4-quinolinecarboxaldehyde, 2,4-pentanedione and anmonia in one step. 
As amines (2-3), the other ammonia source such as ammonium acetate can be used in this reaction. By the use of substituted amines (2-3) (R1=alkyl, aryl) such as alkylamines or arylamine in the above reaction can give the corresponding 1-substituted pyrroles.
In addition, the functional groups at the 1-, 3- or 4-position of the pyrroles prepared above can be converted to a variety of functional groups by the methods known to one skilled in the art.
3. Synthesis of Pyridylpyrroles by Cycloaddition Reaction
The compounds of formula (Ib) or (Ic) can be also prepared by [3+2] cycloaddition as described in Scheme 3. 
(wherein Bt is benztriazole)
In Scheme 3, the compounds of the formula (Ib or Ic) can be prepared by [3+2] cycloaddition of thioamidates ((3-2) or (3-4)) and xcex1,xcex2-unsaturated ketones (Michael acceptor) (3-3). This reaction can be carried out in an inert solvent, such as tetrahydrofuran or dimethylformamide, in the presence of base, preferably sodium hydride. This reaction can be carried out at a temperature in a range from xe2x88x9220 to 150xc2x0 C., preferably 0 to 100xc2x0 C., for 10 minutes to 3 days, usually 3 minutes to 15 hours. The reaction procedures and conditions are described in, for example, Katritzky A. R. et al., Tetrahedron, 1995, 51, 13271.
Thioamides (3-1) can be readily obtained by Mannich condensation of substituted isonicotinoylthioamide, aldehyde and benztriazole according to the literature procedure. Treatment of the thioamides (3-1) with one equivalent of base such as butyllithium or sodium hydride, followed by a reaction with alkyliodide gives thioimidates (3-2). This reaction may be carried out in a reaction inert solvent, for example, tetrahydrofuran, diethylether or methylene chloride, preferably tetrahydrofuran at a temperature in the range from xe2x88x92100 to 50xc2x0 C., preferably xe2x88x9278 to 20xc2x0 C. for 10 minutes to 2 days, usually 30 minutes to 3 hours.
In addition, the thioimidates (3-2) can also be alkylated at the xcex1-position leading to the compound (3-4).
Alternatively, the compounds of formula (I) can be also prepared by a variety of methods known in the art, such as Knorr""s method, Feist""s method, and Hantzsch""s method.
4. Synthesis of Pyridylpyrroles
The compounds of formula (I) can be also prepared from (4-1) by using the method of Stille or Suzuki or by nucleophilic substitution as described in Scheme 4. 
(wherein R1, R2, R3, R4, and R5 are as already defined above; and X is halo such as Cl, Br, or I) The pyrrolyl halides (4-1) can be prepared from the corresponding pyrroles by halogenation known in the art.
The compounds of formula (I) can be prepared by a reaction of compound (4-1) with an appropriate organometallic reagent such as trialkylstannyl, dialkylboronyl, boric acid, in the presence of a catalyst, preferably tetrakis(triphenylphosphine)palladium or bis(triphenylphosphine)palladium(II) chloride, in the inert solvent such as benzene, toluene, xylene, tetrahydrofuran, dioxane, dimethylformamide, preferably dioxane under suitable conditions.
For example, the reaction of phenylboronic acid with compound (4-1) may be carried out in a solvent such as benzene, toluene, dimethoxyethane, dimethylformamide, preferably dimethoxyethane, typically in the presence of a base such as potassium hydroxide, triethylamine, sodium bicarbonate, preferably sodium bicarbonate, or a combination of water and above compounds, preferably water and dimethoxyethane. The catalyst may be selected from those typically employed for the so-called Suzuki reaction (for example, tetrakis(triphenylphosphine)palladium, bis(triphenylphosphine)palladium(II) chloride, or {bis(diphenylphosphino)butane}palladium(II) chloride). The reaction is run at a temperature in the range from 20 to 160xc2x0 C., usually 60 to 130xc2x0 C. for 10 minutes to 5 days, usually 30 minutes to 15 hours. The procedures and conditions to carry out these coupling reactions are known to those in the art.
The compounds of formula (I) can be prepared by a reaction of compound (4-1) with an appropriate alkylamine or cyclic amine such as piperidine, piperazine, or morpholine, without solvent. This reaction proceeds at a temperature in the range from 20 to 250xc2x0 C., preferably 80 to 150xc2x0 C. for 10 minutes to 3 days, usually 30 minutes to 15 hours. This reaction may also be carried out in the presence of a catalyst, preferably tetrakis(triphenylphosphine)palladium or bis(tri-o-tolylphosphine)palladium(II) chloride, in the inert solvent such as benzene, toluene, xylene, tetrahydrofuran, dioxane, dimethylformamide, preferably dioxane under suitable conditions. The procedures and conditions to carry out these coupling reactions are known to those in the art. (for example, Buchwald, S. L. et al., Angew. Chem. Int. Ed. Engl., 1995, 34, 1348.)
As the pyridylpyrrole compounds of this invention may possess at least one asymmetric center, they are capable of occurring in various stereoisomeric forms or configurations. Hence, the compounds can exist in separated (+)- and (xe2x88x92)-optically active forms, as well as in racemic or (xc2x1)-mixtures thereof The present invention includes all such forms within its scope. Individual isomers can be obtained by known methods, such as optically selective reaction or chromatographic separation in the preparation of the final product or its intermediate.
Insofar as the pyridylpyrrole compounds of this invention are basic compounds, they are capable of forming a wide variety of different salts with various inorganic and organic acids.
The acids which are used to prepare the pharmaceutically acceptable acid addition salts of the aforementioned pyridylpyrrole base compounds of this invention of formula (I) are those which form non-toxic acid addition salts, i.e., salts containing pharmaceutically acceptable anions, such as chloride, bromide, iodide, nitrate, sulfate or bisulfate, phosphate or acid phosphate, acetate, lactate, citrate or acid citrate, tartrate or bi-tartrate, succinate, maleate, fu marate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1.1xe2x80x2-methylene-bis-(2-hydroxy-3-naphthoate))salts.
The pharmaceutically acceptable salts of the present invention also include alkali or alkaline earth metal salts such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Quaternary salts obtained from compounds of the invention and C1-4 alkyl halide are also included. The other pharmaceutically acceptable salts which can be used in the present invention are described in J. Pharmacelitical Scienices, 1977, 66, 1-19.
These salts can be prepared by conventional procedures.
The compounds (I) of this invention prepared as mentioned above inhibit inflammatory stimuli-induced cytokines production such as tumor necrosis factor alpha (TNF-xcex1) and interleukine-1xcex2 (IL-1xcex2), and are useful in the treatment or alleviation of various cytokine-mediated diseases such as asthma, arthritis, inflammatory bowel disease (IBD), sepsis, septic shock, rhinitis, inflammation of organs (e.g. hepatitis), AIDS and various inflammatory diseases. Furthermore, the compounds of this invention inhibit inflammatory stimuli-induced synthesis of proteins that regulate adhesion of leukocytes to other leukocytes and to other cell types and have potential use in the treatment of inflammatory and immune disorders such as arthritis and IBD; cardiovascular diseases, psoriasis and transplant rejection.
The pyridylpyrrole compounds of formula (I) of this invention can be administered via either the oral, parenteral or topical routes to mammals. In general, these compounds are most desirably administered to humans in doses ranging from 0.3 mg to 750 mg per day, preferably from 10 mg to 500 mg per day, although variations will necessarily occur depending upon the weight and condition of the subject being treated, the disease state being treated and the particular route of administration chosen. However, for example, a dosage level that is in the range of from 0.06 mg to 2 mg per kg of body weight per day is most desirably employed for the treatment of inflammation.
The compounds (I) of the present invention may be administered alone or in combination with pharmaceutically acceptable carriers or diluents by either of the above routes previously indicated, and such administration can be carried out in single or multiple doses. More particularly, the novel therapeutic agents of the invention can be administered in a wide variety of different dosage forms, i.e., they may be combined with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, troches, hard candies, powders, sprays, creams, salves, suppositories, jellies, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups, and the like. Such carriers include solid diluents or fillers, sterile aqueous media and various nontoxic organic solvents, etc. Moreover, oralpharmaceutical compositions can be suitably sweetened and/or flavored. In general, the therapeutically-effective compounds of this invention are present in such dosage forms at concentration levels ranging 5% to 70% by weight, preferably 10% to 50% by weight.
For oral administration, tablets containing various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate and glycine may be employed along with various disintegrants such as starch and preferably corn, potato or tapioca starch, alginic acid and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in gelatine capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the active ingredient may be combined with various sweetening or flavoring agents, coloring matter or dyes, and, if so desired, emulsifying and/or suspending agents as well, together with such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.
For parenteral administration, solutions of a compound of the present invention in either sesame or peanut oil or in aqueous propylene glycol may be employed. The aqueous solutions should be suitably buffered (preferably pH greater than 8) if necessary and the liquid diluent first rendered isotonic. These aqueous solutions are suitable for intravenous injection purposes. The oily solutions are suitable for intra-articular, intramuscular and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art. Additionally, it is also possible to administer the compounds of the present invention topically when treating inflammatory conditions of the skin and this may preferably be done by way of creams, jellies, gels, pastes, ointments and the like, in accordance with standard pharmaceutical practice.
The ability of the compounds of the formula (I) to inhibit TNFxcex1 biosynthesis and CAMs expression may be demonstrated in vitro by the following procedures.
1. Cells and Cell Culture:
L929 cells are grown in minimum essential medium (MEM) (Gibco BRL NY) supplemented with 10% FCS, 50 U/mL penicillin and 50 xcexcg/mL streptomycin. Human umbilical vein endothelial cells (HUVECs) are obtained from Morinaga and grown in endothelial growth medium (E-GM UV, Kurabou, Japan) supplemented with 10% fetal calf serum (FCS, Biowhitakker, Walkersyille, Md.), 10 ng/mL EGF, 1 xcexcg/mL hydrocortisone, and 1:100 dilution of bovine brain extract (Kurabou, Japan) in 5% CO2 at 37xc2x0 C. A human promyelocytic cell line, HL-60 cells are grown in RPMI-1640 (Nissui Seiyaku, Tokyo, Japan) supplemented with 10% FCS plus penicillin (50 U/mL) and streptomycin (50 xcexcg/mL).
The ability of the compounds of the formula (I) to inhibit TNFxcex1 biosynthesis may be demonstrated in vitro by the following procedure 2.
2. TNFxcex1 Production:
Human peripheral blood mononuclear cells (HPBMNC) are isolated from heparinized human whole blood by Ficoll-Paque (Pharmacia, Sweden) density centrifugation, washed with Caxe2x80x94Mg free phosphate-buffered saline (PBS, Nissui Seiyaku, Tokyo, Japan), suspended in RPMI 1640 containing 10% FCS and plated into 48 well plates (Falcon, Becton Dickinson, N.J.) at 2xc3x97106 cells/well. Monocytes (HBMo) are allowed to adhere to the plate by incubating at 37xc2x0 C. for 1 hour, then the supernatant is aspirated and refilled with fresh RPMI-1640 medium containing 1% FCS.
Test compounds are prepared as 100 mM dimethyl sulfoxide (Me2SO) stock solutions and diluted with media to obtain final testing concentrations. HMo are incubated at 37xc2x0 C. for 4 hours in the presence of LPS (E. coli. 055:B5, Difco, Mich.) of 10 xcexcg/mL with the test compounds in dose ranges of 0.1 xcexcMxcx9c100 xcexcM. The assay is run in a volume of 200 xcexcL/well. Supernatants are subjected to quantitation of TNFxcex1 by an L929 cell cytotoxicity assay. On the day of the experiment, L929 cells are detached by trypsin treatment, washed with MEM and resuspended with 1% FCS-containing MEM. L929 cells (8xc3x97105 cells/well) in a volume of 50 xcexcL are plated into flat-bottomed 96 well plate (Corning, N.Y.) and incubated with 50 xcexcL of serially diluted supernatants in the presence of finally 0.5 xcexcg/mL of actinomycin D (Wako, Japan) at 37xc2x0 C. in 5% CO2 for 18 hours. After incubation, the culture medium is removed and viable cells are stained with 0.2% crystal violet dissolved in 20% ethanol. The cells are washed with tapping water and air-dried at room temperature. Resulting crystal violet is dissolved in 100 xcexcl of 100% methanol and the optical density is determined at 595 nm on a BIOxe2x80x94RAD plate reader (Richmond, Calif.). The concentration of TNFxcex1 is regressed by human recombinant TNFxcex1 (Wako, Japan) set as a standard. Percent inhibition is determined by comparing the absorbance of vehicle treated cells with drug treated cells. Linear regression analysis of the means of the inhibition values are used to determine the IC50s.
Some compounds prepared in the Working Examples as described below were tested by this method, and showed an IC50 value of 100 nM to 10 xcexcM with respect to inhibition of TNFxcex1 biosynthesis.
The ability of the compounds of the formula (I) to inhibit CAMs expression may be demonstrated in vitro by the following procedures 3 and 4.
3. Cell ELISA:
Test compounds are diluted with media to obtain final testing concentrations. HUVECs (1.2xc3x97104/well) grown in flat-bottomed, 96 well, culture plates (Corning, N.Y.) are stimulated with human TNFxcex1 (3 U/mL, Wako, Tokyo, Japan) in the presence or absence of test compounds. Cells are incubated for 6 hours, then washed in PBS, fixed in 4% paraformaldehyde for 15 minutes, washed and stored for 1-3 days at 4xc2x0 C. in PBS.
Adhesion molecules are detected using ELISA. Cells are incubated with a primary antibody to either ICAM-1 (0.5 xcexcg/mL) (BA#3, RandD Systems) or E-selectin (0.5 xcexcg/mL) (BBA#1, RandD Systems). Anti-mouse Ig, peroxidase-linked species-specific F(abxe2x80x2)2 fragment (from sheep) (Amersham; 1:2500 dilution) is used as the second antibody, followed by the addition of peroxidase substrate, o-phenylenediamine. The absorbance of each well is read with a Bio-Rad plate reader at 490 nm, and the background at 655 nm is subtracted. The absorbance of nonstimulated HUVECs is subtracted from the absorbance values of TNFxcex1- stimulated cells. Percent inhibition is determined by comparing the absorbance of vehicle treated cells with drug treated cells. Linear regression analysis of the means of the inhibition values are used to determine the IC50s.
Some compounds prepared in the Working Examples as described below were tested by this method, and showed an IC50 value of 50 nM to 10 xcexcM with respect to inhibition of the CAMs expression.
4. Cell Adhesion Assay:
BL-60 cells are induced to differentiate into granulocyte-like cells by 1.25% Me2SO in RPMI-1640 supplemented with 10% heat-inactivated FCS for 5-6 days. Then cells are incubated with 300 xcexcM of fluorescent dye, 5(6)-carboxyl fluorescein diacetate, for 30 minutes at 37xc2x0 C. and washed three times with Hank""s solution. HUVECs (1.2xc3x97104/well) grown in 96 well plates are simultaneously treated with the test compounds which are diluted with media to obtain final testing concentrations and 30 U/mL TNFxcex1 for 6 hours. Labeled cells (5xc3x97105/well) are added to TNFxcex1- stimulated HUVECs in a final volume of 0.1 mL gently washing four times with warm Hank""s solution, and remaining cells are lysed with 1% Nonidet P-40. The number of adherent cells are determined by measuring the fluorescence intensity using a Fluoroscan II (excitation at 485 nm and emission at 538 nm). Percent inhibition is determined by comparing the fluorescence intensity of vehicle treated cells with drug treated cells. Linear regression analysis of the means of the inhibition values are used to determine the IC50s.
Some compounds prepared in the Working Examples as described below were tested by this method, and showed an IC50 value of 50 nM to 10 xcexcM with respect to inhibition of the adhesion of HL-60 to HUVECs stimulated by TNFxcex1.